Unconjugated hyperbilirubinemia is the most common clinical condition in the newborn period and when severe can result in neurologic injury with long term adverse neurodevelopment sequelae as evidenced by the reemergence of kernicterus in near-term infants subject to early hospital discharge. The pathogenesis of hyperbilirubinemic encephalopathy remains unclear but central to its development is the passage of bilirubin across the blood-brain barrier (BBB) into the central nervous system (CNS). Recent studies suggest that bilirubin is a substrate for the ATP dependent integral plasma membrane efflux pump phosphoglycoprotein or P-gp. P-gp is expressed in abundance on the luminal aspect of brain capillary endothelial cells and limits the brain influx of a variety of lipophilic compounds. We have observed i) that brain bilirubin uptake is significantly increased in adult P-gp deficient transgenie mice, and ii) that P-gp expression (mRNA and protein) is markedly lower in the fetal and neonatal wild type mouse brain as compared with adults. These findings suggest that P-gp plays an important role in preventing the influx of bilirubin into the CNS and that limited P-gp expression in the newborn brain microvasculature may enhance brain bilirubin uptake in this age group. The proposed experiments are designed to test three hypotheses: 1) that bilirubin interacts with and is transported by P-gp; 2) that brain P-gp expression, levels, and function are regulated in a temporal and spatial fashion, and 3) that metabolic inhibition, hypoxia, and/or a combination of hypoxia/ischemia impairs P-gp function. We will use in vitro (radioligand binding and photoaffinity labeling), cellular (mouse brain capillary endothelial cells, and LLC-PK1 cells transfected with the gene for human or mouse P-gp), and in vivo (wild type and P-gp deficient mice) models to characterize the i) interaction between bilirubin and P-gp, ii) the ontogeny and regional expression of CNS P-gp, and iii) the effects of metabolic inhibition on P-gp. Other than our preliminary studies, there is no information regarding the developmental expression of P-gp in the CNS, and only limited data on the interaction between bilirubin and P-gp and the factors that impact P-gp function. Results of the proposed studies will fully test our above stated hypotheses. The information obtained will provide novel insights regarding the role P-gp plays in attenuating brain bilirubin uptake and serve as an impetus towards developing modalities that increase BBB P-gp expression in newborns thereby enhancing protection against neonatal bilirubin neurotoxicity.